Microbes that grow optimally above 60° C. (thermophiles) and 80° C. (hyperthermophiles) populate ecosystems where many biomolecules have reduced stability. To support cellular growth, proteins from such organisms have evolved distinct amino acid compositions and higher thermostability compared to orthologs found in mesophilic organisms that grow at lower temperatures. This latter feature has been exploited for a variety of biotechnological applications.
For example, thermostable DNA polymerases have revolutionized molecular biology, xylanases have made paper processing greener, and oligosaccharide-modifying enzymes have been harnessed for corn syrup production. There is also a great interest in harnessing thermotolerant microbes and their proteins for other industrial processes, such as biomass conversion to bioethanol or biohydrogen. However, no in vivo screens have been developed to help achieve these high-temperature metabolic engineering and synthetic biology goals.
A comparison of the findings from mesophile and hyperthermophile proteomic studies suggests that the lack of high-temperature protein-protein interactions screens may limit the discovery of useful protein complexes. A high-throughput screen for pairwise protein-protein interactions among almost one thousand Pyrococcus horikoshii proteins found only 56 hetero-interactions using a two-hybrid assay implemented at a temperature (37° C.) far below that of its optimal growth of 98° C.
This finding can be contrasted with similar screens for protein complexes in bacteria and yeast under near physiological conditions, which invariably find protein-protein interactions at a frequency that is more than an order of magnitude higher. The creation of an assay that can be used to assess protein complex formation at thermophile growth temperatures would have multiple advantages over available assays in studying natural and engineered proteins. High temperature assays are predicted to be superior at discovering interactions among proteins that require extreme temperatures to adopt their native conformation and among proteins whose interactions weaken as temperature is decreased from the levels where hyperthermophiles grow.
Using split adenylate kinases in protein fragment complementation has been discussed by Nguyen, et al in “Thermostability promotes the cooperative function of split adenylate kinases,” published in Protein Engineering, Design & Selection, Vol. 21, pp. 303-310, 2008.
However, the system discussed in this paper was not in a high-temperature setting, and it is unclear whether the split fragments of adenylate kinases could still function or associate at high temperatures. Nor was the assay tested in a thermophilic organism. Therefore, there is a still need for a protein complementation assay capable of performing at high temperatures.